The objective of this research is to study the structure and replication of mitochondrial DNA from normal and neoplastic cells. Several long-term goals of this program are understanding the function of the mitochondrial genetic system, and understanding why topologically altered mitochondrial DNAs are a distinguishing feature of many malignant cells. The replication origin region will be inserted into a non-conjugated plasmid and this recombinant molecule will be cloned and amplified in Escherichia coli. A detailed map of the restriction endonuclease sites in this region will be constructed. This map will be used as a basis for the nucleotide sequence analysis of the origin of replication, thereby deducing and comparing with several other systems the structural features of the origin. Experiments will be directed toward understanding aspects of the replicative mechanism, and the mechanism of complex mitochondrial DNA formation. In particular, we will investigate whether circular dimers arise by a replicative or by a recombinational mechanism. In addition, we propose to explore the use of a plasmid derived from colicinogenic factor E1 as a manipulable model system to study the formation of circular dimers in vivo.